1. Field of the Invention
This invention relates to the high resolution replication of specimens, including both biological and non-biological specimens, and the use of such replicas in high resolution microscopic analyses. In particular aspects, the invention concerns the preparation and use of replicas of biological samples such as cells, tissues and the like, as well as non-biological materials obtained therefrom, in connection with freeze-fracture, freeze-etching and related microscopic techniques. Moreover, the invention contemplates the applicability of the disclosed replica technology in connection with a wide variety of microscopic techniques including transmission electron microscopy (TEM), scanning electron microscopy (SEM), scanning tunneling microscopy, and even atomic force microscopy.
2. Description of the Related Art
More and more, the use of microscopic analysis in both biological and materials sciences has become an extremely important adjunct to investigational as well as quality control analyses. Electron microscope replication techniques were devised during the 1940s by workers in the materials sciences, particularly metallurgy, for the examination of surface topography (see, for example, Mahl, H. (1040), Z. Techn. Phys.. 21:17, and Gerould, C. H. (1947), J. Appl. Phys., 18:333). While these early techniques have now become largely obsolete, they nevertheless demonstrated the particularly usefulness of surface examination in the material sciences.
In the biological sciences, surface examination has been equally important. Structural analyses of biological substances, including biological samples such as cell components as well as biochemicals such as enzymes, receptors, etc., have played a major role over the years in obtaining a spatial resolution of structural relationships. This has, as one might well imagine, led to numerous breakthroughs in cell and molecular biological investigations, not the least of which has been a delineation of cell surface receptor structure/function relationship.
Transmission electron microscope technology has certain inherent limitations. For example, in transmission electron microscopy, when one desires to view a specimen directly, it must be sufficiently thin to allow the transmission of electrons. Certain very small specimens are inherently electron-transparent and may be viewed directly by a variety of means, including shadowing. Most specimens, however, are too thick to allow electrons to be transmitted, and a variety of thinning techniques have been developed allowing internal structure to be studied. However, transmission analyses, per se, do not allow the observation of surfaces of structures and rather are limited to the investigation of internal structure.
As alluded to above, a variety of techniques have been developed over the years which approach the problem of surface analyses of structures, as opposed to analyses of internal structure. One such approach is referred to generally as the replication of specimens or "replica" technique. In these techniques, a thin electron-transparent layer, which faithfully molds the surface contours, is prepared and then stripped from the specimen and analyzed microscopically. Contrast in such replicas results from the contour of the surface being replicated, as well as the use of shadowing techniques which employ the deposition of a metal or otherwise electron-opaque film on the surface. For a very helpful text which covers most all of the general aspects of specimen replication, see Willison et al. (1980), Practical Methods in Electron Microscopy, Volume 8: Replica, Shadowing and Freeze-Etching Techniques, North-Holland Publishing Company, ed. Audrey Glauert, pp. 1-101, incorporated herein by reference.
The principal disadvantages associated with replica techniques generally concerns the fact that the replica is not always able to faithfully form a contour of the surface being replicated. While techniques have been devised for the replication of virtually every type of surface (see, e.g., Bradley, D. E. (1965), Replica in Shadowing Techniques, In: Techniques for Electron Microscopy, Second Edition, D. H Kay, ed., Blackwell Oxford), problems nevertheless arise with regular surfaces, as well as surfaces where a high degree of resolution is required to view the particular structure. The problem of resolution is particular acute where one desires to observe the macromolecular morphology or tertiary structure of enzymes, nucleic acids, structural proteins, glycoproteins, polysaccharides, or the like.
In general, the currently available replicating techniques are inadequate for viewing structures below 15 to 20 angstroms. This lack of resolving capability greatly reduces the usefulness and versatility of the replication in connection with a wide variety of applications. For example, many important quality control applications in the field of computer technology (e.g., in chip manufacturing) would be much better served were there the capability available for viewing extremely minute structures on chips, e.g., in determining their uniformity of manufacture. Moreover, in the biological sciences, many important cell-surface structures are somewhat smaller than 15 to 20 or even smaller than 10 angstroms, thus, requiring greater resolving capabilities than currently available by the replica technique.
Accordingly, there is currently a need for improved techniques in the replica technology area, a need which has prior to now gone unaddressed. In particular, there is a need for improvements in replication technology which can be applied using essentially existing hardware, both in terms of the replica-forming hardware as well as the microscope hardware, without a requirement for expensive, complex or hard-to-use techniques. Moreover, there is a particular need for techniques that can be applied in virtually any laboratory across the country without a requirement for extensive training or expertise.